Assessment of Reagents for Selenocysteine Conjugation and the Stability of Selenocysteine Adducts

Selenocysteine Conjugation

Traditional antibody-drug conjugates (ADCs) are heterogeneous mixtures with poor pharmacokinetic properties and lower efficacy than homogeneous ADCs. Therefore, there is an urgent need to study site-specific antibody conjugation to solve this problem. Selenocysteine, an unnatural amino acid (UAA), and modified antibodies have been developed that allow site-specific antibody conjugation, producing homogeneous ADCs.

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In this study, scientists studied several electrophilic functional groups that react with selenocysteine with high efficiency and determined the stability of the resulting conjugates. On this basis, it was further proved that allene functionality can generate stable, site-specific SELENOMAB conjugates with drugs, fluorophores, and contrast agents. It also offers another functional group that, together with heteroaryl sulfones, can be used for dual labeling of ADCs.

The Stability Assessment of Selenocysteine Adducts 11-15

In this article, researchers respectively evaluated the stability of selenocysteine adducts 11-15 (Fig. 1) under different conditions, including weak acidity, alkalinity, oxidation, the presence of glutathione, and human plasma.

Fig. 1. Schematic diagram of the structure of the selenocysteine adducts 11-15. (Pedzisa L, et al., 2016)

• Under mildly acidic conditions, Sec-ODA 11, Sec-BTA 12, and Sec-allene (Sec-ALL) 15 adducts were stable with minor degradation (Fig. 2, blue bars). In comparison, Sec-MAL 13 and Sec-iodoacemide (Sec-IAM) 14 adducts degraded significantly.
• Under basic and oxidizing conditions, analogous trends were observed, where ≥ 60% of Sec-ODA 11, Sec-BTA 12, and Sec-ALL 15 remained after 72 h while less than 60% of Sec-MAL 13 and Sec-IAM 14 (Fig. 2, red and green bars).
• Under the presence of glutathione conditions, similarly, Sec-ODA 11, Sec-BTA 12, and Sec-ALL 15 were the most stable, with ≥ 70% remaining, while Sec-MAL 13 (32% remaining) and Sec-IAM 14 (60% remaining) were considerably less stable.

Fig. 2. Stability evaluation for selenocysteine conjugates. (Pedzisa L, et al., 2016)

• In human plasma at 37 °C, Sec-ODA 11 was the least stable (t_1/2 < 24 h) (Fig. 3, blue line), and Sec-BTA 12 was more stable than Sec-ODA 11. Sec-IAM 14 was the next most stable (t_1/2 = 77 h), and Sec-ALL 15 was the most stable (t_1/2 = 139 h).

Fig. 3. Stability evaluation of Sec-conjugates in human plasma. (Pedzisa L, et al., 2016)

SELENOMAB-Fluorescein Conjugate Characterization

Based on the above results and the excellent robustness of Sec-ALL 15 from this stability profile, researchers further synthesized SELENOMAB-fluorescein 23 and 24 (SFC-ALL and SFC-ODA) conjugates using Sec-ALL 15 and evaluated their antigenic activity and stability in human plasma.

• Antigen Binding Activity

Flow cytometry was used to investigate the ability of SELENOMAB-fluorescein conjugates to bind to HER2 receptors on the surface of SK-BR-3 breast cancer cells using flow cytometry.

SK-BR-3 cells incubated with allene-based 23 (SFC-ALL) and those incubated with oxadiazole-based 24 (SFC-ODA) SELENOMAB-fluorescein conjugates had a shift in fluorescence relative to the background, and the SELENOMAB-fluorescein conjugates only bound to HER2 receptors, which shows that fluorescein-conjugated SELENOMABs retain HER2-binding activity (Fig. 4).

Fig. 4. Fluorescein conjugated SELENOMABs retain HER2-binding activity. (Pedzisa L, et al., 2016)

• Plasma Stability

The conjugates were incubated in human plasma at 37°C for 3 days. Aliquots were collected at 0, 4, 8, 12, 24, 48, and 72 h and analyzed by SDS-PAGE.

While the sulfone-based conjugate 24 (SFC-ODA) was moderately stable with a half-life of about 12 h, the allene-based conjugate 23 (SFC-ALL) was very stable with a half-life of over 28 days (Fig. 5).

Fig. 5. Allene and sulfone-based SELENOMAB-fluorescein conjugate stability in human plasma. (Pedzisa L, et al., 2016)

Reference

1. Pedzisa L, Li X, Rader C, et al. Assessment of reagents for selenocysteine conjugation and the stability of selenocysteine adducts. Org Biomol Chem. 2016,14(22):5141-7.

For Research Use Only. NOT FOR CLINICAL USE.